International Journal of Molecular Sciences Review Role of Aiolos and Ikaros in the Antitumor and Immunomodulatory Activity of IMiDs in Multiple Myeloma: Better to Lose Than to Find Them Marco Cippitelli 1,* , Helena Stabile 1, Andrea Kosta 1, Sara Petillo 1, Angela Gismondi 1, Angela Santoni 1,2 and Cinzia Fionda 1,* 1 Department of Molecular Medicine, Istituto Pasteur-Fondazione Cenci Bolognetti, Sapienza University of Rome, 00185 Rome, Italy; [email protected] (H.S.); [email protected] (A.K.); [email protected] (S.P.); [email protected] (A.G.); [email protected] (A.S.) 2 IRCCS Neuromed–Instituto Neurologico Mediterraneo, 86077 Pozzilli, Italy * Correspondence: [email protected] (M.C.); cinzia.fi[email protected] (C.F.); Tel.: +39-06-4434-0632 (M.C. & C.F.) Abstract: The Ikaros zing-finger family transcription factors (IKZF TFs) are important regulators of lymphocyte development and differentiation and are also highly expressed in B cell malignancies, including Multiple Myeloma (MM), where they are required for cancer cell growth and survival. Moreover, IKZF TFs negatively control the functional properties of many immune cells. Thus, the targeting of these proteins has relevant therapeutic implications in cancer. Indeed, accumulating evidence demonstrated that downregulation of Ikaros and Aiolos, two members of the IKZF family, in malignant plasma cells as well as in adaptative and innate lymphocytes, is key for the anti-myeloma activity of Immunomodulatory drugs (IMiDs). This review is focused on IKZF TF-related pathways Citation: Cippitelli, M.; Stabile, H.; Kosta, A.; Petillo, S.; Gismondi, A.; in MM. In particular, we will address how the depletion of IKZF TFs exerts cytotoxic effects on Santoni, A.; Fionda, C. Role of Aiolos MM cells, by reducing their survival and proliferation, and concomitantly potentiates the antitumor and Ikaros in the Antitumor and immune response, thus contributing to therapeutic efficacy of IMiDs, a cornerstone in the treatment Immunomodulatory Activity of of this neoplasia. IMiDs in Multiple Myeloma: Better to Lose Than to Find Them. Int. J. Mol. Keywords: IKZF; Ikaros; Aiolos; IMiDs; Lenalidomide; Multiple Myeloma Sci. 2021, 22, 1103. https://doi.org/ 10.3390/ijms22031103 Academic Editor: 1. Introduction Tomasz Wilanowski The Ikaros zinc-finger (IKZF) protein family consists of five Kruppel-like transcrip- Received: 28 December 2020 tion factors (TFs) (named Ikaros, Helios, Aiolos, Eos and Pegasus or IKZF1-IKZF5) with Accepted: 19 January 2021 Published: 22 January 2021 a well-documented role in lymphocyte development and differentiation [1,2]. Lack of Ikaros proteins from the hemopoietic system leads to an impaired production of B and Publisher’s Note: MDPI stays neutral T lymphocytes and Natural Killer (NK) cells [3]. Consistently, altered function of these with regard to jurisdictional claims in proteins has been associated with autoimmunity and cancer [4–6]. Indeed, Ikaros proteins published maps and institutional affil- act as tumor suppressors in different types of leukemia (where their function is inhibited), iations. whereas they are overexpressed in other malignancies (where they are necessary) to sustain the survival and proliferation of cancer cells, such as in Multiple Myeloma (MM) [7,8]. Multiple Myeloma is an incurable cancer in which malignant plasma cells (PCs) grow and accumulate within bone marrow (BM), causing end-organ dysfunctions and morbidity. MM is a very complex and heterogeneous disease which mainly affects elderly people with Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. a median age of 65 years [9–11]. In most cases, MM is preceded by a premalignant condi- This article is an open access article tion, named monoclonal gammopathy of undetermined significance (MGUS). The disease distributed under the terms and conceivably evolves upon accumulation of genetic mutations and deep alterations of the conditions of the Creative Commons BM microenvironment. Increasing evidence highlights how the compromised functionality Attribution (CC BY) license (https:// of the immune system is crucial for disease progression [12]. A number of mechanisms creativecommons.org/licenses/by/ contribute to prevent anti-myeloma immune response, such as the defective function of 4.0/). dendritic cells (DC) and effector lymphocytes and the expansion of immunosuppressive Int. J. Mol. Sci. 2021, 22, 1103. https://doi.org/10.3390/ijms22031103 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 1103 2 of 13 cells (e.g., myeloid-derived suppressor cells and regulatory T cells, Treg) [13]. Accord- ingly, some therapeutic approaches to overcome immune dysfunctions have already been approved for MM treatment and several others are currently under investigation. A cornerstone in the current MM therapy is represented by immunomodulatory drugs (IMiDs, e.g., thalidomide, lenalidomide and pomalidomide). Lenalidomide is used in the treatment of newly diagnosed patients, in the post-transplant maintenance therapy and in the relapsed/refractory setting, while pomalidomide is used only in the relapsed/refractory disease [14]. IMiDs exert multiple effects on different cellular components present in tumor microenvironment accounting for their profound immunostimulatory properties and direct antitumor activity. Ten years ago, Ito T. and colleagues demonstrated that the cellular target of these drugs is Cereblon (CRBN), a ubiquitous protein which functions as a substrate re- ceptor for the Cullin-4-RING Ubiquitin Ligase (CLR4) complex also including DDB1, ROC1 and CUL4 [15]. Binding of IMiDs to CRBN alters its substrate specificity, thus disrupting or promoting the recruitment and ubiquitination of many proteins [16]. A number of studies described the capability of lenalidomide and pomalidomide to induce CRBN-dependent degradation of Ikaros and Aiolos in different cell types initiating multiple downstream effects, which were shown to be responsible for the antitumor and immunomodulatory properties of these drugs [7,17,18]. Here, we will summarize evidence from the current literature about the complex role of IKZF TFs in regulating the survival and susceptibility of MM cells to immune-mediated attack but also their function in immune effector cells. Moreover, we will discuss how the inhibition of these critical TFs highly contributes to the clinical activity of IMiDs in MM. 2. IKZF TFs: Molecular Structure and Transcriptional Activity All IKZF TFs share the same structural properties. They are characterized by functional domains composed of zinc-finger motifs: an N-terminal DNA-binding domain to bind the consensus sequence A/GGGAA, and two C-terminal dimerization domains to interact with other IKZF proteins. Moreover, shorter IKZF variants can be generated by alternative splicing. These isoforms conserve C-terminal dimerization domains but have a different number of N-terminal zinc fingers. IKZF TFs lacking N-terminal zinc fingers do not bind DNA and function as dominant negative (DN) [19]. IKZF proteins can act both as repressors and as activators of gene transcription [20]. Upon binding DNA, they trigger epigenetic regulation and chromatin-remodeling through the direct interaction with two types of remodeling factors, the corepressor (e.g., the nucle- osome remodeling and deacetylase complex, NuRD, or C-terminal binding protein, CtBP) and coactivator proteins (Switch/Sucrose-Nonfermentable chromatin-remodeling complex, SWI/SNF). IKZF TFs can repress gene transcription by histone deacetylase (HDAC) depen- dent and independent mechanisms. They can promote hypo-acetylation of core histones at promoter sites via direct interaction with several corepressor proteins (e.g., NuRD complex ATPase, Mi-2β, Sin3A and Sin3B) which bind HDAC-containing complexes [21]; in addi- tion, they can silence gene expression by recruiting the co-repressor CtBP, which in turn represses transcription initiation through the interactions with the general transcription factors TFIIB and TBP in the transcription pre-initiation complex [22,23]. In a different way, the capability of IKZF TFs to activate gene transcription is due to the formation of activated enhancers and super-enhancers possibly through their interaction with the gene activating complex SWI/SNF [24]. IKZF proteins have different expression patterns. Eos and Pegasus are widely dis- tributed in different tissues, while Ikaros, Helios and Aiolos are mainly expressed in immune system cells, and in addition Ikaros and Helios can be found in some non-immune cells, such as erythrocytes. Importantly, several studies demonstrated a role for these proteins in cancer. Impaired functionality of Ikaros, often caused by IKZF DN isoform overexpression, deletions or mutations, has been associated with the development of leukemia (e.g., B and T Acute Lymphoblastic Leukemia, ALL, Acute and Chronic Myeloid Leukemia) [25], indicating a tumor suppressive activity for this transcription factor. In this Int. J. Mol. Sci. 2021, 22, 1103 3 of 13 regard, long-term studies of IMiDs maintenance therapy in MM patients have revealed increased frequencies of hematologic second primary malignancies (SPMs), including B-ALL [26,27], suggesting the involvement of therapy-induced modulation of these TFs. In a different way, Ikaros and Aiolos are overexpressed and fully functional in other hematological and solid cancers. For instance, both proteins are highly expressed in MM cells at different stages of disease progression from normal